Nörotransmitterlerin tayini için hplc'de metot geliştirilmesi ve metot validasyonu

In this study, two methods were developed for the determination of the neurotransmitters (gamma aminobutyric acid (GABA), glutamate, serotonin and noradrenaline) and the melatonin hormone in cerebrospinal fluid samples from the hippocampus, amygdala and raphe nuclei of the rat brain by using high-performance liquid chromatography (HPLC) with fluorescence detection and a C18 reversed-phase column. Samples were collected by using microdialysis technique. In the first method, pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion was performed for the simultaneous detection of GABA and glutamate. The derivatives were separated using isocratic elution and detected fluorimetrically at the excitation and the emission wavelengths of 420 and 480 nm, respectively. The second method involves the simultaneous detection of melatonin, serotonin and noradrenaline without derivatization step. The compounds were separated using isocratic elution and detected fluorimetrically at the excitation and the emission wavelengths of 280 and 340 nm, respectively. The methods were validated. The performance of the methods was evaluated in terms of linearity, method detection limit, limit of quantitation, recovery and precision. Standard addition method was used to obtain calibration curves. The method detection limit values were found as 28 ppb for GABA, 9 ppb for glutamate, 0.5 ppb for melatonin, 1.2 ppb for serotonin and 2.7 ppb for noradrenaline. The limit of quantitation values were found as 92 ppb for GABA, 30 ppb for glutamate, 1.7 ppb for melatonin, 3.8 ppb for serotonin and 8.8 ppb for noradrenaline. The average recoveries changed between 103 % and 118 % for GABA, 87 % and 99 % for glutamate, 88 % and 121 % for melatonin, 76 % and 117 % for serotonin and 72 % and 105 % for noradrenaline. The percentage relative standard deviation (RSD) values ranged from 0.78 to 1.80 for GABA, from 0.80 to 1.67 for glutamate, from 0.31 to 2.18 for melatonin, from 0.84 to 5.05 for serotonin and from 0.44 to 8.24 for noradrenaline. Measurement uncertainties of all compounds were calculated by applying 'bottom up' method. The calculated percentage relative uncertainty values for the compounds changed between 10.1 and 16.7. The major sources of the uncertainty budget were calibration curves, stock solution and recovery. Keywords: Neurotransmitter, GABA, Glutamate, Serotonin, Noradrenaline, Melatonin, Cerebrospinal Fluid, Microdialysis, HPLC, Fluorescence Detection, Method Validation, Uncertainty Budget